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This ELISA kit applies to the in vitro quantitative determination of Rat CRH concentrations in serum, plasma and other biological fluids.
This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with Rat CRH. During the reaction, Rat CRH in the sample or standard competes with a fixed amount of Rat CRH on the solid phase supporter for sites on the Biotinylated Detection Ab specific to Rat CRH. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Rat CRH in the samples is then determined by comparing the OD of the samples to the standard curve.
|Detection Range||0.31—20 ng/mL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Rat CRH in samples. No significant cross-reactivity or interference between Rat CRH and analogues was observed.
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat CRH were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat CRH were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||10.00||3.70||4.49||9.09||3.70||4.88|
The recovery of Rat CRH spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||86-98||91|
|Cell culture media (n=5)||89-99||94|
Samples were spiked with high concentrations of Rat CRH and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
An unopened kit can be stored at 2-8°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20°C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 μL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 μL||-20°C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||2-8°C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||2-8°C(shading light)|
|Stop Solution||1 vial, 10 mL||2-8°C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution.
The volume of reagents in partial shipments is a little more than the volume marked on the label, please use accurate measuring equipment instead of directly pouring into the vial(s).
1. Add 50μL standard or sample to the wells, immediately add 50μL Biotinylated Detection Ab working solution to each well. Incubate for 45 min at 37°C
2. Aspirate and wash the plate for 3 times
3. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times
4. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C
5. Add 50μL Stop Solution
6. Read the plate at 450nm immediately. Calculation of the results